1. Field of the Invention
The present invention relates to methods for monitoring expression of a plurality of genes in filamentous fungal cells. The present invention also relates to substrates and computer readable media for monitoring expression of a plurality of genes in filamentous fungal cells.
2. Description of the Related Art
Microarray technology is increasingly becoming the method of choice for the quantitative and simultaneous analysis of the expression levels of many thousands of genes. Microarray analyses typically follow the steps of gene selection, microarray synthesis, sample preparation, array hybridization, detection, and data analysis (Watson et al., 1998, Current Opinion in Biotechnology 9: 609-614).
PCR-amplified coding sequences of genomic DNA are particularly useful in microarrays for obtaining global expression profiles where the genome of the organism has been fully sequenced. Chu et al., 1998, Science 282: 699-705, disclose the use of microarrays containing PCR-amplified genomic coding sequences for determining the temporal expression of Saccharomyces cerevisiae genes during sporulation.
For other organisms whose genomes have not been sequenced, global expression profiles may be obtained with arraying (1) random genomic DNA segments or clones (e.g., from a genomic DNA library); (2) random cDNA clones (e.g., from one or more cDNA libraries) that are uncharacterized at the DNA sequence level; (3) EST clones that have been sequenced and partially characterized with respect to putative identification and function; or (4) cDNA clones that are enriched for differentially expressed sequences (Diatchenko et al., 1996, Proc Natl. Acad. Sci. USA 93: 6025-6030).
However, there are disadvantages with using random genomic or cDNA clones from organisms whose genomes have not been fully sequenced. These disadvantages include (1) more than one gene may be represented on a single clone; (2) no gene(s) may be encoded on a single clone; (3) extensive characterization and DNA sequencing is required to follow-up array spots that appear interesting; and (4) duplicity, multiplicity, and redundancy add to the follow-up work.
Expressed sequenced tags (ESTs) are partial cDNA sequences of expressed genes. Simply stated, an EST is a segment of a sequence from a cDNA clone that corresponds to the mRNA of a specific gene. The use of sequenced ESTs in microarrays compared to genomic clones or random cDNA clones provides several advantages especially for organisms whose genomes have not been sequenced. First, redundancy is eliminated because one spot on an array equals one gene or open reading frame. Second, since sequence information is available, redundancy and follow-up characterization is minimized. Third, EST microarrays can be organized based on function of the gene products to facilitate analysis of the results (e.g., ESTs encoding enzymes from the same metabolic pathway can be arranged or grouped accordingly).
Ruan et al., 1998, The Plant Journal 15: 821-833, disclose the use of microarrays containing Arabidopsis thaliana EST sequences for determining the temporal expression of Arabidopsis thaliana genes in root, leaf, and two stages of floral development.
Iyer et al., 1999, Science 283; 83-87, disclose the use of microarrays containing human EST sequences for determining the temporal expression of human fibroblast cells in response to serum.
Diatchenko et al., 1996, supra, disclose a method called suppression subtractive hybridization (SSH) for generating differentially regulated or tissue-specific cDNA probes and libraries. Yang et al., 1999, Nucleic Acids Research 27: 1517-1523, describe combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Porkka and Visakorpi, 2001, Journal of Pathology 193: 73-79, disclose detection of differentially expressed genes in prostrate cancer by combining SSH and cDNA library arrays.
Hayward et al., 2000, Molecular Microbiology 35: 6-14, disclose shotgun DNA microarrays and stage-specific gene expression in Plasmodium falciparum malaria.
WO 2000/56762 discloses methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing filamentous fungal expressed sequenced tags.
Filamentous fungi are increasingly being used as host microorganisms for the industrial production of enzymes and other proteins whether endogenous or heterogenous to the microorganisms. There is a need in the art to provide methods for monitoring the global expression of genes from filamentous fungal cells to improve the production potential of these microorganisms and to identify new genes whose products have industrial applicability.
It is an object of the present invention to provide alternative methods for monitoring expression of a plurality of genes in filamentous fungal cells.